Project Summary It is generally thought that DNA replication evolved twice independently in Bacteria and Archaea/Eukarya, as the principle components of the replication machinery such as the replicative helicase and the DNA polymerases are not evolutionarily related in the two branches of life. In mammals, chromosome replication error or insufficient correction of the replication error is a major cause of cancers. Therefore, understanding the molecular mechanism of eukaryotic DNA replication is important not only for fundamental biology but also for understanding and developing treatment for human diseases. The replisome progression complex (RPC) is composed of replicative helicase the Cdc45-Mcm2-7-GINS (CMG) complex, the leading strand DNA polymerase epsilon, the lagging strand DNA polymerase delta, the polymerase alpha-primase, and over a dozen additional protein factors. Because of its sheer size and dynamic nature, very little is known about the eukaryotic replisome architecture. However, recent advance in cryo-EM methodology, along with the most recent in vitro reconstitution of the leading strand and the lagging strand DNA synthesis, has made it feasible to tackle this challenge. In a preliminary study, the PI's lab has used cryo-EM to determine an atomic model of the 11-protein CMG helicase, and mapped a few key components of the replisome, including Pol epsilon, Ctf4, and Pol alpha. Because the individual Mcm proteins are bi-lobed and elongated, the Mcm2-7 is a double ring-like structure with the C-terminal AAA+ motor ring stacked on top of the N-tier ring. EM work has shown that the leading strand polymerase epsilon binds to the C-tier motor ring, whereas the Pol alpha-primase is recruited by Ctf4 to the N-tier ring side of the CMG helicase. Therefore, the two polymerases reside on the opposite side of the helicase, resulting to a profoundly asymmetric replisome architecture. Building on that success, the PI proposes to continue the cryo- EM structural and biochemical investigation, on how CMG interacts with the forked DNA, and how CMG scaffolds the replisome. The PI has teamed up with an expert replication biochemist; together they are well poised to elucidate the structure and function of the eukaryotic replisome. The proposed research is significant because the replisome plays a central role in cellular growth, and dysregulation of replication can lead to uncontrolled proliferation and tumorigenesis.